The major emphasis of this research project deals with the mechanisms of substrate-induced homolytic cleavage of the Co-C Female bond in B12-coenzyme catalysis. Model studies with methylcobalamin demonstrate that homolysis of the Co-C bond occurs in the transfer of methyl-groups to CrII, SnII as well as to PtII/PtIV complexes. Also, we have shown that homolysis occurs in a methyl-transfer to coenzyme M (ethanethiol sulfonic acid) by a B12-dependent transmethylase purified from cell-extracts of Methanosarcina barkeri grown on methanol as sole carbon source. Using a variety of spectroscopic techniques, we have obtained preliminary evidence that the formation of a "charge-transfer" complex between substrate and the corrin macrocycle precedes homolysis of the Co-C bond. Therefore, complexation of the substrate with the corrin-ring occurs, rather than direct attack on the Co-C bond. These new results indicate that electrophiles can react with B12-coenzymes either by direct attack at the Co-C bond (i.e. SE2 mechanism) (e.g. HgII, PbIV, TlIII and PdII); or by reacting as oxidants through complexation with the corrin ring. (e.g. IrIV, PtII-X-PtIV and FeIII). The rate-determining step for the latter reaction appears to involve a one-electron transfer from the corrin ring to the complexed electrophile. Since homolytic cleavage of the Co-C bond occurs in a number of B12-enzyme catalyzed reactions, and since this mechanism is not fully understood, we intend to expand our study of reaction intermediates in B12-dependent methyl-transfer to platinum salts, and to coenzyme M by the transmethylase which we have purified from M. barkeri. A similar B12-dependent transmethylase is implicated in C1-metabolism in Clostridium thermoacticum (1). This organism synthesizes acetyl CoA from carbon monoxide and coenzyme A. Using 13C NMR in a wide bore 360 MHz NMR spectrometer we intend to perform a comparative study of autotrophic metabolism on C1 compounds, by using 13C enriched substrates for M. barkeri (i.e. 13CH3OH, 13HCOOH and 13CO) and for C. thermoaceticum (13CO and 13CO2). The role of B12 in whole cells, and at the enzyme level, will be studied in an effort to delineate the metabolic pathways for the autotrophic growth of anaerobes on C1 compounds.